Expression of many genes is limited by the ability of RNA polymerase to complete polymerization of up to a million nucleotides, making elongation to emerge next to initiation as a major regulatory step in gene expression. Several accessory protein factors that allow RNA polymerase to overcome this limitation and become "elongation-proficient have been described. The focus of this proposal, the bacterial protein RfaH, is a regulator of several long virulence and fertility operons, where it preferentially increases the expression of distal genes. We have demonstrated that RfaH binds to its recruitment sequence, ops, exposed on the surface of the RNA polymerase paused at an ops site during elongation. Following its recruitment, RfaH stimulates transcription downstream of an ops site by enhancing elongation rate and suppressing pausing. However, RfaH only modestly inhibits termination. The detailed mechanism of RfaH action, described as "antitermination', remains obscure except for the fact that it is different from those of other antiterminators such as lambdaN and lambdaQ, which have profound effects on both elongation and termination. Both the recruitment mode and the effect of RfaH on elongation are unique, thus insights into the RfaH mechanism will contribute to the general understanding of the regulation of transcript elongation in bacteria and also in eukaryotes, where RfaH homologs are implicated in elongation control and localize to the actively transcribed sites. In this proposal, we will use a combination of biochemical, genetic, and biophysical approaches to address several aspects of RfaH action. The first goal of this project is to elucidate the molecular mechanism by which RfaH affects elongation thousands of nucleotides downstream of its recruitment site. The central mechanistic question to be answered is whether RfaH travels with the elongating RNA polymerase or if it causes a conformational change in the RNA polymerase that persists for thousands of nucleotide addition steps after RfaH dissociates from the complex. The second goal of this project is to determine how universal is this mechanism by finding out whether RfaH affects transcription similarly at all sites or is targeted to a particular set of regulatory signals. The third goal of this project is to map interactions between RfaH and the transcription elongation complex, thus placing RfaH mechanism in its structural context. RfaH controls the expression of the secreted molecules, components of the cell wall, antibiotics, virulence factors, and proteins required for the mobilization of transmissible plasmids. Proposed studies will therefore positively impact research in several areas of bacterial biology and evolution, such as synthesis of extracytoplasmic components, bacterial virulence, lateral gene transfer, and emergence of pathogens. [unreadable] [unreadable] [unreadable]